Fish Physiologyand Biochemistryvol. 6 no. 6 pp 333-340 (1989) Kugler Publications, Amsterdam/Berkeley
Myosin heavy chain synthesis in white trunk muscle of cod (Gadus morhua) fed different ration sizes Alexandra von der Decken I and Einar Lied 2 I The Wenner-Gren Institute for Experimental Biology, University o f Stockholm, S-106 91 Stockholm, Sweden, and 2Institute o f Nutrition, Directorate of Fisheries, N-5013 Nygaardstangen/Bergen, Norway Keywords: cod, Gadus morhua, myosin heavy chain, ration size, epaxial muscle, immunotechnique, mRNA-cDNA hybridization
Abstract
In this study the expression of myosin heavy chain was investigated at the messenger RNA and protein level of the muscle ribosome. Cod (Gadus morhua) weighing 80 g were fed for 70 days either ad libitum or with rations consisting of 75070, 50~ or 25~ of the ad libitum food intake. Protein and RNA contents of the ribosome from the white trunk muscle decreased with the diminished ration sizes. Myosin heavy chain content relative to total ribosome protein increased with the 50% ration size but fell again to the ad libitum level at 2507o. Expressed relative to RNA, a decrease in the protein content occurred in parallel with the decreasing ration size. The amount of protein/total muscle homogenate fell with diminished ration sizes, and that of myosin heavy chain/mg o f protein remained unchanged with a slight increase at the 25~ food energy intake. The messenger RNA for myosin heavy chain (relative to poly(A)+messenger RNA) was increased in response to the decreased ration size but was decreased when calculated per g wet weight of the muscle. The changes in messenger RNA for myosin heavy chain were less pronounced than those of the protein itself. The levels of gene transcription and translation for myosin heavy chain were affected to a lesser extent by the low food intake than the synthesis of total RNA and protein. Immunological methods and messenger RNA hybridization to cloned DNA for myosin heavy chain permitted the precise determinations of the events taking place during a food-energy supply shortage. Translation appeared to diminish prior to changes in messenger RNA concentrations.
Introduction
In warm-blooded animals, the efficiency of food utilization is reflected in the protein synthesis activity of skeletal muscle (Omstedt and v o n d e r Decken 1972, 1974; von der Decken et al. 1979). In fish a similar correlation is found (Smith 1981; Lied et al. 1982). The relationship between food intake and growth rate has been well-documented in cod
(Hawkins et al. 1985). Growth is a function of both rates of synthesis and degradation. To achieve optimal growth of the muscle tissue ad libitum food intake exceeds the optimal requirements of cod (Lied et al. 1985). A ration size of 75~ is sufficient for optimal growth, whilst a 25070 ration suffices only for the maintenance level of the fish and decreases markedly the protein synthesis activity in vitro. White muscle of cod shows a high efficiency
Abbreviations: mRNA, messenger RNA; mRNP, messengerribonucleoproteinparticles; cDNA, cloned DNA; poly(A)+mRNA, polyadenylated messenger RNA.
334 o f protein retention and increased protein synthesis with increased growth rates (Houlihan et al. 1988). The tissue comprises 10-12~ protein, the protein composition being o f sarco'plasmic and myofibrillar type ( A n d o et al. 1986), the latter being necessary for normal muscle function. The myosin heavy chain protein is representative o f the myofibrillar proteins, being the largest in size and weight. Polyclonal antibodies have been raised against the isolated protein (Lied and von der Decken 1985). In this. study the effects o f decreasing the ration size fed to cod were examined. In contrast to previous studies, this investigation was directed towards measurements o f a specific protein. The myosin heavy chain synthesis and content were assayed by means o f the E L I S A technique and c D N A - m R N A hybridization was used to analyze for myosin heavy chain m R N A in muscle. The contribution of the present study to the knowledge o f the sequence o f evefits in the expression o f myosin heavy chain both at the m R N A and the protein concentration level provides a basis for the understanding o f the myosin changes which occur during restricted food supply.
Materials and methods Materials
The sources o f materials have been described previously (Lied and von der Decken 1985). All other chemicals o f the highest purity available were supplied by Sigma Chemical Co. (St. Louis, MO) and Serva (Heidelberg, FRG).
Table 1. Composition of the experimental diet
Ingredients
g/kg wet weight
Saithe ( Pollachius virens) fillet L Krill (Meganyctiphanes norvegica)1 Capelin (Mallotus vitrosus) oil I Dextrin from wheat starch I Vitamin mixturez Mineral mixture 3 Guar gum (binding agent) I
665 167 52 91 12 3 10
The estimated metabolizable energy of the diet was 4400 kcal/kg dry wt. and protein content 47~ of the total energy. The values used in the calculation were: per g of protein 3.9 kcal, fat 8.0 kcal, carbohydrate 3.0 kcal; Icommercially available; -'composition of the vitamin mixture (rag vitamin/kg vitamin mixture): thiamine-HCI 167; riboflavin 333; pyridoxine 167; Ca-pantothenate 667; niacin 2500; folic acid 83; ascorbic acid 667; vitamin A 16,400 IU; vitamin D 2680 IU; u-tocopherol acetate 60,000 IU; 3commercial standard mineral mixture used for poultry and swine containing (g/kg): phosphorus 60; calcium 240; sodium 60; magnesium 10; iron 2; manganese 2; zinc 2.5; copper 0.4; iodine 0.075; selenium 0.008. energy intake was varied. All groups received food once a day. F o o d was withdrawn f r o m the ad libiturn g r o u p when the fish were satiated. The ration size was then calculated and the other g r o u p s were given 75070, 50~ or 2507o that o f the ad libitum level. At the end o f the feeding experiment all fish were killed by a blow to the head and weighed. The epaxial muscle o f the white type was dissected, and slices o f the muscle were wrapped in aluminium foil, immediately frozen between two solid blocks o f CO 2 and stored at - 8 0 ~ ( k u n d and v o n d e r Decken 1980; Lied et al. 1982).
Preparation o f r i b o s o m e s Fish and diets
C o d were hatched at the A q u a c u l t u r e Station Austevoll, on the west coast of N o r w a y , and were maintained in 1.7 m 3 water tanks supplied with running sea water at 7 - 8 ~ The mean salinity was 3.5%. Fish weighing between 75 to 82 g were selected from a stock o f i m m a t u r e fish and were r a n d o m ly divided into 4 groups with 40 fish in each. The feeding experiment lasted for 70 days. The g r o u p s were all fed the same diet (Table 1) except that the
The m e t h o d o f preparing ribosomes has been described previously (Lied et al. 1982). In short, 2.5 g o f muscle were thawed in medium (0.25 M sucrose, 0.1 M Tris-HCl buffer pH 7.8 at 25~ 0.28 M KCI, 5 mM MgSO4), h o m o g e n i z e d and centrifuged for 10 min at 1,200 x gmax" F r o m the supernatant fractions ribosomes were recovered by centrifugation for 2h at 165,000 • (Beckman L2-65 B centrifuge, 50 Ti angle rotor, Beckman Instruments, Palo Alto, CA).
335 Sodium acetate-ethanol precipitated ribosomes were prepared by suspending the ribosome pellet in 0.01 M Na-phosphate buffer 0.45 M NaCI, pH 7 to give a concentration of 1 mg protein/ml. Sodium acetate (final concentration of 4%) and two volumes of ethanol were then added. The suspension was kept at - 2 0 ~ for one day, centrifuged for 10 min at 10,000 x gay and the pellet was suspended in the above Na-phosphate-NaC1 buffer to give a protein concentration of 1 mg/ml.
Immunoassay The ELISA procedure was applied in a competitive inhibition analysis using myosin heavy chain specific antibodies (Engvall and Perlmann 1972; Lied and yon der Decken 1985).
Isolation o f poly(A) +m R N A Muscle tissue (0.3 g) was homogenized in 1 ml of medium (0.1 M Tris-HCl buffer pH 7.8 measured at 25~ 0.25 M KCI and 5 mM MgSO 4) containing 10 mM vanadyl nucleoside (Meinkoth and Wahl 1984). After centrifugation for 10 min at 1,200 x gmax the supernatant was incubated with 300 #g/ml of proteinase K for 1 h at 25~ Guanidinium HCI (final concentration of 4 M) was added and then RNA was isolated (Arrand 1985). P o l y ( A ) + m R N A was then isolated by the oligo d(T) method (Bantle et al. 1976).
c D N A - m R N A dot hybridizations The relative level of the myosin specific mRNA was measured by dot hybridization using GreenScreen Plus membranes (NEN Research Products, Boston, MA) and following the procedure described by Anderson and Young (1985). The genomic DNA for myosin heavy chain was isolated and sequenced from Caenorhabditis elegans by Karn et al. (1983). It was radioactively labelled by nick translation using the Amersham Kit (Amersham International Ltd, Amersham, UK) with (3H)dCTP (spec. activi-
ty 30 Ci/mmol) as a precursor (Arrand 1985). The specific activity was 9 x 10 6 dpm/p.g of DNA. Hybridization was followed by washing. The dots were then cut out and the radioactivity was measured in a scintillation spectrometer (Beckman LS 3801, Beckman Instruments, Palo Alto, CA).
Analysis Proteins of the ribosome fraction and total muscle homogenate were analyzed by the Coomassie brilliant blue method (Bradford 1976) using bovine serum albumin as a standard. DNA of the whole muscle was analyzed by the fluorometric method using salmon DNA as a standard (Setaro and Morley 1976). RNA of the ribosome fraction was analyzed by extracting the ribosomes in 0.4 M HCIO 4 for 18 min at 70~ The absorbance of the soluble fraction was measured at 260 nm and RNA calculated on basis of 34.2 absorbance units/mg of RNA.
Polyacrylamide gel electrophoresis Electrophoresis of proteins was performed in gradient gels between 7% and 15% polyacrylamide, containing 0.1% sodium dodecylsulfate, using the slab gel apparatus (Hames 1983).
Statistical analysis Radioactivity values for the hybridization were compared by one-way analysis of va'riance. Slopes of the linear function of radioactivity vs. RNA concentration were calculated by linear regression. All other results were calculated using one-way analysis of variance and Newman Keuls' test for multiple sample comparison (Snedecor and Cochran 1980).
Results
Growth rates and muscle composition The fish were fed the specified ration sizes for 70
336 T a b l e 2. G r o w t h a n d liver w e i g h t s ( L W ) after feedir~g d e c r e a s i n g r a t i o n sizes I B o d y wt(g)
Percent weight gain
kW ( % o f b o d y wt):'
R a t i o n size
Initial
Final
Ad libitum 3
81.2 _+2.2
147.2 _+5.4 a
80.6
11.5 _+0.5 a
75% 50o;0
74.5 _+2. I 78.9 _+ 2.2
123.2 __+4.0 h I 12.5 _+3.3 ~
65.4 42.6
10.6 _+0.4 a 8.4 _+0.3 b
25%
79.5 _+2. I
87.2 _+2.6 d
9.7
6.2 _+0.3':
IValues are given as m e a n _+ SEM of 40 fish in each g r o u p ; m e a n s w i t h i n one c o l u m n not f o l l o w e d by the s a m e s u p e r s c r i p t letter are significantly d i f f e r e n t (p < 0.05); :initial liver ,,,,'eight ( % o f b o d y weight) was 7.0_+0.4; 3energy i n t a k e of a d l i b i t u m fed fish 35 k c a l / l ~ g / d a y .
T a b l e 3. C o n t e n t o f r i b o s o m a l p r o t e i n , R N A a n d D N A [ e x p r e s s e d / g `.vet `.,,'eight o f muscle] after feeding d e c r e a s i n g r a t i o n sizes I
Protein (mg)
R N A (mg)
Ribosome
NAEP
Ribosome
NAEP
R a t i o n size
pellet:
r i b o s o m e s :~
pellet"
ribosomes 3
D N A (mg)
Ad libitum 4
7507o
21.54 a _+ 1.23 16.'12 I' _+ 1.74
1.23 a _+0.15 1.37 a _+_0.31
1.14 a _+ 0.13 0.79 h _+0.09
0.46 a _+0.09 0.37 a,c ___0.01
0.698 a _+0.017 0.706 a _+0.061
50%
11.34 b _ 1.98
0.92 ax + 0.32
0.65 b _+0.06
0.25 bx _+0.02
0.568 a _-+0.036
259
7.42': _+_1.42
0.52 b.c + 0.13
0.55 b _+0.05
0.20 b _+0.03
0.671 a _+0.040
IValues are gi`. en as m e a n _+ SEM o 1 6 fish per g r o u p ; m e a n s within one c o l u m n not f o l l o w e d by the s a m e superscript letter are significantly d i f f e r e n t (p < 0.05); 2complete r i b o s o m e pellet; ~ N A E P = s o d i u m a c e t a t e - e t h a n o l p r e c i p i t a t e d r i b o s o m e s [to r e m o v e n o n r i b o s o m e - b o u n d proteins (see M a t e r i a l s a n d M e t h o d s ) ] ; 4see T a b l e 2.
days. The changes in body weight and liver wet weight are summarized in Table 2. At diminished ration sizes the growth rate decreased as did the liver'weight (as a % of body weight). Ribosomal RNA, together with the proteins associated with the ribosome fraction, decreased significantly with the diminished ration sizes (Table 3). A decrease in RNA/g wet weight was correlated with the diminished ration sizes and showed significant differences to the ad libitum fed fish. No significant changes in D N A / g wet weight were noted. Previous analyses by polyacrylamide gel electrophoresis of the ribosome fraction have shown a high content of myosin and actin associated with that fraction (Rosenlund et al. 1983). Suspension of the ribosome pellet followed by precipitation with sodium acetate-ethanol removed most of the myosin and actin, leaving behind the proteins which were more strongly bound to the ribosomes (Fig. 1).
Myosin heavy chain content
Myosin heavy chain protein of the total and sodiumacetate-ethanol precipitated ribosomes was assayed using the ELISA technique (Table 4). When calculated per mg of protein, the myosin heavy chain content of the 75~ and 25~ ration size was not significantly different from the ad libitum feeding. With the 50% ration size, however, the content was significantly elevated indicating a rise in myosin heavy chain content relative to the other muscle proteins. With reference to RNA no differences between ration sizes were seen. A decreasing trend in the content of myosin heavy chain/mg of tissue DNA was observed which when expressed as per g of wet weight muscle became more pronounced and significant with the decreasing ration sizes. The myosin heavy chain of the sodium acetateethanol precipitated ribosomes was not significantly different between groups. With decreasing ration sizes the amount of pro-
337 (A)+mRNA added to the filters and the mRNA hybridized to the cloned DNA coding for myosin heavy chain (Fig. 2). The amount of mRNA for myosin heavy chain relative to total poly(A) +mRNA was lowest in the ad libitum fed fish and increased in the 50% and 25% ration sizes. The latter was significantly elevated over ad libitum fed fish by a factor of 1.64 (Table 6). As shown in Table 3, the total amount of R N A / g wet weight of muscle was 2.07- fold higher in the fish fed ad libitum than those fed the 25% ration size. Thus, a recalculation per g wet weight of muscle showed the highest level of mRNA for myosin heavy chain in the ad libitum fed fish and a decrease in the 50% and 25% ration size indicating a decrease in total mRNA for myosin heavy chain with decreasing ration size.
Fig. I. Polyacrylamide gel electrophoresis in sodium dodecylsul-
fate of protein derived from sodium acetate-ethanol precipitated ribosomes (lane 2 and 3), total ribosomes (lane 4 and 5), and molecular ',,,eight marker (lane I and 6) of 66K, 43K, 25K and 12.5K. 15/ag and 7.5/.Lg of protein ',','ere applied to the gels. The arrow indicates the position of myosin heavy chain. The gel was stained v,.ith Coomassie brilliant blue-R250 which has low affinity to the basic ribosomal proteins (molecular weight below 30K).
tein/g wet ,.,,,eight of tissue homogenate fell (Table 5) and myosin/mg of protein remained constant. Relative concentrations o f myosin heavy chain mRNA
There was a linear relationship between the poly-
Discussion
The content of myosin heavy chain present in the total ribosome fraction confirmed the diminished overall protein synthesis activity obtained in ration size experiments and demonstrated by a different approach (Lied et al. 1985). The results indicate that myosin heavy chain production was lowered at a slower rate than the decrease in protein content/g wet weight of tissue. If myosin heavy chain is representative of the myofibrillar proteins, the results may be interpreted as a saving of the contractile elements to be utilized for the vital physical activity of the fish. The major part bf muscle used in the present study belonged to the white type. White muscle is
Table 4. Content of myosin heavy chain in muscle ribosomes expressed as m g / m g protein (A), m g / m g RNA (B), m g / m g tissue DNA (C) and mg/g v,'et weight (D)
A
B
C
Ralion size
Total ribosomes2
NAEP ribosomes 3
Total ribosomes
NAEP ribosomes
A d libitum ~ 75% 50~ 25~
0,330 ~ +_0.030 0,210a+_0.024 0,504 b _+0.072 0.277 a _+0.035
0.059 0.053 0.033 0.033
5.90_+0.29 3.07_+0.49 5.57+_ 1.71 3.66+_0.53
0.145_+0.024 10.20a_+1.13 0.156+0.033 5.12ax___1.32 0.128+0.030 8.57a_+2.40 0.091 _+0.025 3.07b'c_+0.61
+_0.015 _+0.008 +_0.003 +_0.005
Total ribosomes
D NAEP ribosomes
Total ribosomes
NAEP ribosomes
0.118'~_+0.014 0.123"_+0.014 0.099~ _+0.013 0.050a _+0.010
7.13a_+0.85 3.45t'-0.55 4.54a.r 2.07bx-+0.48
0.081a-+0.012 0.080a_+0.012 0.049a'b-+0.010 0.037a +_0.009
IValues are given as mean ___ SEM of 6 fish per group; means within a column not followed by the same superscript letter are significantly different (p
Myosin heavy chain synthesis in white trunk muscle of cod (Gadus morhua) fed different ration sizes Alexandra von der Decken I and Einar Lied 2 I The Wenner-Gren Institute for Experimental Biology, University o f Stockholm, S-106 91 Stockholm, Sweden, and 2Institute o f Nutrition, Directorate of Fisheries, N-5013 Nygaardstangen/Bergen, Norway Keywords: cod, Gadus morhua, myosin heavy chain, ration size, epaxial muscle, immunotechnique, mRNA-cDNA hybridization
Abstract
In this study the expression of myosin heavy chain was investigated at the messenger RNA and protein level of the muscle ribosome. Cod (Gadus morhua) weighing 80 g were fed for 70 days either ad libitum or with rations consisting of 75070, 50~ or 25~ of the ad libitum food intake. Protein and RNA contents of the ribosome from the white trunk muscle decreased with the diminished ration sizes. Myosin heavy chain content relative to total ribosome protein increased with the 50% ration size but fell again to the ad libitum level at 2507o. Expressed relative to RNA, a decrease in the protein content occurred in parallel with the decreasing ration size. The amount of protein/total muscle homogenate fell with diminished ration sizes, and that of myosin heavy chain/mg o f protein remained unchanged with a slight increase at the 25~ food energy intake. The messenger RNA for myosin heavy chain (relative to poly(A)+messenger RNA) was increased in response to the decreased ration size but was decreased when calculated per g wet weight of the muscle. The changes in messenger RNA for myosin heavy chain were less pronounced than those of the protein itself. The levels of gene transcription and translation for myosin heavy chain were affected to a lesser extent by the low food intake than the synthesis of total RNA and protein. Immunological methods and messenger RNA hybridization to cloned DNA for myosin heavy chain permitted the precise determinations of the events taking place during a food-energy supply shortage. Translation appeared to diminish prior to changes in messenger RNA concentrations.
Introduction
In warm-blooded animals, the efficiency of food utilization is reflected in the protein synthesis activity of skeletal muscle (Omstedt and v o n d e r Decken 1972, 1974; von der Decken et al. 1979). In fish a similar correlation is found (Smith 1981; Lied et al. 1982). The relationship between food intake and growth rate has been well-documented in cod
(Hawkins et al. 1985). Growth is a function of both rates of synthesis and degradation. To achieve optimal growth of the muscle tissue ad libitum food intake exceeds the optimal requirements of cod (Lied et al. 1985). A ration size of 75~ is sufficient for optimal growth, whilst a 25070 ration suffices only for the maintenance level of the fish and decreases markedly the protein synthesis activity in vitro. White muscle of cod shows a high efficiency
Abbreviations: mRNA, messenger RNA; mRNP, messengerribonucleoproteinparticles; cDNA, cloned DNA; poly(A)+mRNA, polyadenylated messenger RNA.
334 o f protein retention and increased protein synthesis with increased growth rates (Houlihan et al. 1988). The tissue comprises 10-12~ protein, the protein composition being o f sarco'plasmic and myofibrillar type ( A n d o et al. 1986), the latter being necessary for normal muscle function. The myosin heavy chain protein is representative o f the myofibrillar proteins, being the largest in size and weight. Polyclonal antibodies have been raised against the isolated protein (Lied and von der Decken 1985). In this. study the effects o f decreasing the ration size fed to cod were examined. In contrast to previous studies, this investigation was directed towards measurements o f a specific protein. The myosin heavy chain synthesis and content were assayed by means o f the E L I S A technique and c D N A - m R N A hybridization was used to analyze for myosin heavy chain m R N A in muscle. The contribution of the present study to the knowledge o f the sequence o f evefits in the expression o f myosin heavy chain both at the m R N A and the protein concentration level provides a basis for the understanding o f the myosin changes which occur during restricted food supply.
Materials and methods Materials
The sources o f materials have been described previously (Lied and von der Decken 1985). All other chemicals o f the highest purity available were supplied by Sigma Chemical Co. (St. Louis, MO) and Serva (Heidelberg, FRG).
Table 1. Composition of the experimental diet
Ingredients
g/kg wet weight
Saithe ( Pollachius virens) fillet L Krill (Meganyctiphanes norvegica)1 Capelin (Mallotus vitrosus) oil I Dextrin from wheat starch I Vitamin mixturez Mineral mixture 3 Guar gum (binding agent) I
665 167 52 91 12 3 10
The estimated metabolizable energy of the diet was 4400 kcal/kg dry wt. and protein content 47~ of the total energy. The values used in the calculation were: per g of protein 3.9 kcal, fat 8.0 kcal, carbohydrate 3.0 kcal; Icommercially available; -'composition of the vitamin mixture (rag vitamin/kg vitamin mixture): thiamine-HCI 167; riboflavin 333; pyridoxine 167; Ca-pantothenate 667; niacin 2500; folic acid 83; ascorbic acid 667; vitamin A 16,400 IU; vitamin D 2680 IU; u-tocopherol acetate 60,000 IU; 3commercial standard mineral mixture used for poultry and swine containing (g/kg): phosphorus 60; calcium 240; sodium 60; magnesium 10; iron 2; manganese 2; zinc 2.5; copper 0.4; iodine 0.075; selenium 0.008. energy intake was varied. All groups received food once a day. F o o d was withdrawn f r o m the ad libiturn g r o u p when the fish were satiated. The ration size was then calculated and the other g r o u p s were given 75070, 50~ or 2507o that o f the ad libitum level. At the end o f the feeding experiment all fish were killed by a blow to the head and weighed. The epaxial muscle o f the white type was dissected, and slices o f the muscle were wrapped in aluminium foil, immediately frozen between two solid blocks o f CO 2 and stored at - 8 0 ~ ( k u n d and v o n d e r Decken 1980; Lied et al. 1982).
Preparation o f r i b o s o m e s Fish and diets
C o d were hatched at the A q u a c u l t u r e Station Austevoll, on the west coast of N o r w a y , and were maintained in 1.7 m 3 water tanks supplied with running sea water at 7 - 8 ~ The mean salinity was 3.5%. Fish weighing between 75 to 82 g were selected from a stock o f i m m a t u r e fish and were r a n d o m ly divided into 4 groups with 40 fish in each. The feeding experiment lasted for 70 days. The g r o u p s were all fed the same diet (Table 1) except that the
The m e t h o d o f preparing ribosomes has been described previously (Lied et al. 1982). In short, 2.5 g o f muscle were thawed in medium (0.25 M sucrose, 0.1 M Tris-HCl buffer pH 7.8 at 25~ 0.28 M KCI, 5 mM MgSO4), h o m o g e n i z e d and centrifuged for 10 min at 1,200 x gmax" F r o m the supernatant fractions ribosomes were recovered by centrifugation for 2h at 165,000 • (Beckman L2-65 B centrifuge, 50 Ti angle rotor, Beckman Instruments, Palo Alto, CA).
335 Sodium acetate-ethanol precipitated ribosomes were prepared by suspending the ribosome pellet in 0.01 M Na-phosphate buffer 0.45 M NaCI, pH 7 to give a concentration of 1 mg protein/ml. Sodium acetate (final concentration of 4%) and two volumes of ethanol were then added. The suspension was kept at - 2 0 ~ for one day, centrifuged for 10 min at 10,000 x gay and the pellet was suspended in the above Na-phosphate-NaC1 buffer to give a protein concentration of 1 mg/ml.
Immunoassay The ELISA procedure was applied in a competitive inhibition analysis using myosin heavy chain specific antibodies (Engvall and Perlmann 1972; Lied and yon der Decken 1985).
Isolation o f poly(A) +m R N A Muscle tissue (0.3 g) was homogenized in 1 ml of medium (0.1 M Tris-HCl buffer pH 7.8 measured at 25~ 0.25 M KCI and 5 mM MgSO 4) containing 10 mM vanadyl nucleoside (Meinkoth and Wahl 1984). After centrifugation for 10 min at 1,200 x gmax the supernatant was incubated with 300 #g/ml of proteinase K for 1 h at 25~ Guanidinium HCI (final concentration of 4 M) was added and then RNA was isolated (Arrand 1985). P o l y ( A ) + m R N A was then isolated by the oligo d(T) method (Bantle et al. 1976).
c D N A - m R N A dot hybridizations The relative level of the myosin specific mRNA was measured by dot hybridization using GreenScreen Plus membranes (NEN Research Products, Boston, MA) and following the procedure described by Anderson and Young (1985). The genomic DNA for myosin heavy chain was isolated and sequenced from Caenorhabditis elegans by Karn et al. (1983). It was radioactively labelled by nick translation using the Amersham Kit (Amersham International Ltd, Amersham, UK) with (3H)dCTP (spec. activi-
ty 30 Ci/mmol) as a precursor (Arrand 1985). The specific activity was 9 x 10 6 dpm/p.g of DNA. Hybridization was followed by washing. The dots were then cut out and the radioactivity was measured in a scintillation spectrometer (Beckman LS 3801, Beckman Instruments, Palo Alto, CA).
Analysis Proteins of the ribosome fraction and total muscle homogenate were analyzed by the Coomassie brilliant blue method (Bradford 1976) using bovine serum albumin as a standard. DNA of the whole muscle was analyzed by the fluorometric method using salmon DNA as a standard (Setaro and Morley 1976). RNA of the ribosome fraction was analyzed by extracting the ribosomes in 0.4 M HCIO 4 for 18 min at 70~ The absorbance of the soluble fraction was measured at 260 nm and RNA calculated on basis of 34.2 absorbance units/mg of RNA.
Polyacrylamide gel electrophoresis Electrophoresis of proteins was performed in gradient gels between 7% and 15% polyacrylamide, containing 0.1% sodium dodecylsulfate, using the slab gel apparatus (Hames 1983).
Statistical analysis Radioactivity values for the hybridization were compared by one-way analysis of va'riance. Slopes of the linear function of radioactivity vs. RNA concentration were calculated by linear regression. All other results were calculated using one-way analysis of variance and Newman Keuls' test for multiple sample comparison (Snedecor and Cochran 1980).
Results
Growth rates and muscle composition The fish were fed the specified ration sizes for 70
336 T a b l e 2. G r o w t h a n d liver w e i g h t s ( L W ) after feedir~g d e c r e a s i n g r a t i o n sizes I B o d y wt(g)
Percent weight gain
kW ( % o f b o d y wt):'
R a t i o n size
Initial
Final
Ad libitum 3
81.2 _+2.2
147.2 _+5.4 a
80.6
11.5 _+0.5 a
75% 50o;0
74.5 _+2. I 78.9 _+ 2.2
123.2 __+4.0 h I 12.5 _+3.3 ~
65.4 42.6
10.6 _+0.4 a 8.4 _+0.3 b
25%
79.5 _+2. I
87.2 _+2.6 d
9.7
6.2 _+0.3':
IValues are given as m e a n _+ SEM of 40 fish in each g r o u p ; m e a n s w i t h i n one c o l u m n not f o l l o w e d by the s a m e s u p e r s c r i p t letter are significantly d i f f e r e n t (p < 0.05); :initial liver ,,,,'eight ( % o f b o d y weight) was 7.0_+0.4; 3energy i n t a k e of a d l i b i t u m fed fish 35 k c a l / l ~ g / d a y .
T a b l e 3. C o n t e n t o f r i b o s o m a l p r o t e i n , R N A a n d D N A [ e x p r e s s e d / g `.vet `.,,'eight o f muscle] after feeding d e c r e a s i n g r a t i o n sizes I
Protein (mg)
R N A (mg)
Ribosome
NAEP
Ribosome
NAEP
R a t i o n size
pellet:
r i b o s o m e s :~
pellet"
ribosomes 3
D N A (mg)
Ad libitum 4
7507o
21.54 a _+ 1.23 16.'12 I' _+ 1.74
1.23 a _+0.15 1.37 a _+_0.31
1.14 a _+ 0.13 0.79 h _+0.09
0.46 a _+0.09 0.37 a,c ___0.01
0.698 a _+0.017 0.706 a _+0.061
50%
11.34 b _ 1.98
0.92 ax + 0.32
0.65 b _+0.06
0.25 bx _+0.02
0.568 a _-+0.036
259
7.42': _+_1.42
0.52 b.c + 0.13
0.55 b _+0.05
0.20 b _+0.03
0.671 a _+0.040
IValues are gi`. en as m e a n _+ SEM o 1 6 fish per g r o u p ; m e a n s within one c o l u m n not f o l l o w e d by the s a m e superscript letter are significantly d i f f e r e n t (p < 0.05); 2complete r i b o s o m e pellet; ~ N A E P = s o d i u m a c e t a t e - e t h a n o l p r e c i p i t a t e d r i b o s o m e s [to r e m o v e n o n r i b o s o m e - b o u n d proteins (see M a t e r i a l s a n d M e t h o d s ) ] ; 4see T a b l e 2.
days. The changes in body weight and liver wet weight are summarized in Table 2. At diminished ration sizes the growth rate decreased as did the liver'weight (as a % of body weight). Ribosomal RNA, together with the proteins associated with the ribosome fraction, decreased significantly with the diminished ration sizes (Table 3). A decrease in RNA/g wet weight was correlated with the diminished ration sizes and showed significant differences to the ad libitum fed fish. No significant changes in D N A / g wet weight were noted. Previous analyses by polyacrylamide gel electrophoresis of the ribosome fraction have shown a high content of myosin and actin associated with that fraction (Rosenlund et al. 1983). Suspension of the ribosome pellet followed by precipitation with sodium acetate-ethanol removed most of the myosin and actin, leaving behind the proteins which were more strongly bound to the ribosomes (Fig. 1).
Myosin heavy chain content
Myosin heavy chain protein of the total and sodiumacetate-ethanol precipitated ribosomes was assayed using the ELISA technique (Table 4). When calculated per mg of protein, the myosin heavy chain content of the 75~ and 25~ ration size was not significantly different from the ad libitum feeding. With the 50% ration size, however, the content was significantly elevated indicating a rise in myosin heavy chain content relative to the other muscle proteins. With reference to RNA no differences between ration sizes were seen. A decreasing trend in the content of myosin heavy chain/mg of tissue DNA was observed which when expressed as per g of wet weight muscle became more pronounced and significant with the decreasing ration sizes. The myosin heavy chain of the sodium acetateethanol precipitated ribosomes was not significantly different between groups. With decreasing ration sizes the amount of pro-
337 (A)+mRNA added to the filters and the mRNA hybridized to the cloned DNA coding for myosin heavy chain (Fig. 2). The amount of mRNA for myosin heavy chain relative to total poly(A) +mRNA was lowest in the ad libitum fed fish and increased in the 50% and 25% ration sizes. The latter was significantly elevated over ad libitum fed fish by a factor of 1.64 (Table 6). As shown in Table 3, the total amount of R N A / g wet weight of muscle was 2.07- fold higher in the fish fed ad libitum than those fed the 25% ration size. Thus, a recalculation per g wet weight of muscle showed the highest level of mRNA for myosin heavy chain in the ad libitum fed fish and a decrease in the 50% and 25% ration size indicating a decrease in total mRNA for myosin heavy chain with decreasing ration size.
Fig. I. Polyacrylamide gel electrophoresis in sodium dodecylsul-
fate of protein derived from sodium acetate-ethanol precipitated ribosomes (lane 2 and 3), total ribosomes (lane 4 and 5), and molecular ',,,eight marker (lane I and 6) of 66K, 43K, 25K and 12.5K. 15/ag and 7.5/.Lg of protein ',','ere applied to the gels. The arrow indicates the position of myosin heavy chain. The gel was stained v,.ith Coomassie brilliant blue-R250 which has low affinity to the basic ribosomal proteins (molecular weight below 30K).
tein/g wet ,.,,,eight of tissue homogenate fell (Table 5) and myosin/mg of protein remained constant. Relative concentrations o f myosin heavy chain mRNA
There was a linear relationship between the poly-
Discussion
The content of myosin heavy chain present in the total ribosome fraction confirmed the diminished overall protein synthesis activity obtained in ration size experiments and demonstrated by a different approach (Lied et al. 1985). The results indicate that myosin heavy chain production was lowered at a slower rate than the decrease in protein content/g wet weight of tissue. If myosin heavy chain is representative of the myofibrillar proteins, the results may be interpreted as a saving of the contractile elements to be utilized for the vital physical activity of the fish. The major part bf muscle used in the present study belonged to the white type. White muscle is
Table 4. Content of myosin heavy chain in muscle ribosomes expressed as m g / m g protein (A), m g / m g RNA (B), m g / m g tissue DNA (C) and mg/g v,'et weight (D)
A
B
C
Ralion size
Total ribosomes2
NAEP ribosomes 3
Total ribosomes
NAEP ribosomes
A d libitum ~ 75% 50~ 25~
0,330 ~ +_0.030 0,210a+_0.024 0,504 b _+0.072 0.277 a _+0.035
0.059 0.053 0.033 0.033
5.90_+0.29 3.07_+0.49 5.57+_ 1.71 3.66+_0.53
0.145_+0.024 10.20a_+1.13 0.156+0.033 5.12ax___1.32 0.128+0.030 8.57a_+2.40 0.091 _+0.025 3.07b'c_+0.61
+_0.015 _+0.008 +_0.003 +_0.005
Total ribosomes
D NAEP ribosomes
Total ribosomes
NAEP ribosomes
0.118'~_+0.014 0.123"_+0.014 0.099~ _+0.013 0.050a _+0.010
7.13a_+0.85 3.45t'-0.55 4.54a.r 2.07bx-+0.48
0.081a-+0.012 0.080a_+0.012 0.049a'b-+0.010 0.037a +_0.009
IValues are given as mean ___ SEM of 6 fish per group; means within a column not followed by the same superscript letter are significantly different (p
